Hexanoic acid, 3,5,5-trimethyl-, 1,1'-[2-ethyl-2-[[(3,5,5-trimethyl-1-oxohexyl)oxy]methyl]-1,3-propanediyl] ester

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-18 to 2013-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The test was performed prior to the adoption of the standard in vitro and in chemico method and their subsequent incoporation into the standard testing requirements.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

Ear thickness measurements were not taken.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 2013-06-18 To: 2013-07-02

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary test: 100 %
Main test: 25 and 50 % v/v in acetone/olive oil 4:1 and 100 %

No. of animals per dose:

Preliminary screening test: 1 female/dose
Main test: 4 females/dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Test item was used undiluted and freshly prepared as a solution at the concentrations of 25 and 50 % in acetone/olive oil 4:1
- Irritation: Mouse treated with undiluted test item for three consecutive days (Days 1, 2 and 3) showed no signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness. Based on this information the undiluted test item and the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 µL of test material at concentrations of 0 (vehicle control), 25, 50 and 100 % to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). On Day 6, all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. Lymph node cells were washed with PBS and precipitated with 5 % (w/v) trichloroacetic acid (TCA) for radioactive material at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid (Optiphase ‘Trisafe’). 3HTdR incorporation was measured by β -scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No data

Positive control results:

Stimulation index for α-hexylcinnamaldehyde at 25 % v/v in acetone/olive oil 4:1 was 7.33 and classified as a sensitiser.

Key result

Parameter:

SI

Value:

1.26

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.28

Test group / Remarks:

50%

Parameter:

SI

Value:

1.48

Test group / Remarks:

100%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per group for vehicle, 25, 50 and 100 % were 8967.55, 11288.98, 11444.45 and 13314.84, respectively. DPM per node for vehicle, 25, 50 and 100 % were 1120.94, 1411.12, 1430.56 and 1664.36, respectively.

Table 7.4.1/1: Results of skin sensitization

Concentration (% v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	8967.55	1120.94	na	na
25	11288.98	1411.12	1.26	Negative
50	11444.45	1430.56	1.28	Negative
100	13314.84	1664.36	1.48	Negative

dpm = Disintegrations per minute

a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total

number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

Clinical observations and mortality data: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions,the substance is not classified as sensitizing to the skin according to CLP Regulation (EC) No. 1272/2008

Executive summary:

Test Guidance

OECD Guideline 429 and EC Method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Method and materials

Groups of CBA/Ca (CBA/CaOlaHsd) mice (4 females/dose) were topically applied with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50 % or 25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. On Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3HTdR). Five hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3HTdR. The results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study using one female mouse at the concentration of 100 %.

Results

No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. DPM per group for vehicle, 25, 50 and 100 % were 8967.55, 11288.98, 11444.45 and 13314.84, respectively. DPM per node for vehicle, 25, 50 and 100 % were 1120.94, 1411.12, 1430.56 and 1664.36, respectively. Stimulation index for 25, 50 and 100 % were 1.26, 1.28 and 1.48, respectively. Positive control (α-hexylcinnamaldehyde, 25%) exhibited evidence of sensitisation indicating the validity of the study. In this study, the substance is not a skin sensitizer in mice.

Conclusion

Under these test conditions, the substance is not classified as sensitising to the skin according to the CLP Regulation (EC)No. 1272/2008).

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

One reliable study is available. In this study (Sanders, 2013) performed under GLP according to OECD TG 429 and EC Method B.42, groups of CBA/Ca mice (4 females/dose) were topically applied with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50 % or 25 % v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone. On Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3HTdR). Five hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3HTdR. The results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study using one female mouse at the concentration of 100 %. No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. DPM per group for vehicle, 25, 50 and 100 % were 8967.55, 11288.98, 11444.45 and 13314.84, respectively. DPM per node for vehicle, 25, 50 and 100 % were 1120.94, 1411.12, 1430.56 and 1664.36, respectively. Stimulation index for 25, 50 and 100 % were 1.26, 1.28 and 1.48, respectively. Positive control (α-hexylcinnamaldehyde, 25%) exhibited evidence of sensitisation indicating the validity of the study. In this study, the substance is not a skin sensitizer in mice.

Supporting data are also available. Read-across to the toxicological properties of fatty acid polyols (Fatty acids, C5-9, esters with pentaerythritol (EC 270-290-3, CAS 68424-30-6) and Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (EC 234-392-1, CAS 11138-60-6)) and their analogues is applicable based on the similarity in structure and physico-chemical properties.

The justification for read-across is presented in Section 13 Assessment reports- Read-across justification.

In a supporting study, a Guinea Pig Maximisation Test of Skin Sensitisation was performed with CAS No. 11138-60-6 (Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate) according to OECD Guideline 406 (Blanset, 1997). 20 male and female Dunkin-Harley guinea pigs were treated with the test substance and compared with 10 negative control animals. The sensitivity of the animal strain was tested using Hexylcinnamic aldehyde (HCA) as positive control substance. A 5% dilution of the test substance in propylene glycol was used for intradermal induction and 100% used for epidermal induction of the scapular region on days 1 and 8. 14 days after the last induction treatment, all animals were challenged epicutaneously with the undiluted test substance. 24 h after challenge one out of twenty animals showed a skin reaction compared to 2 out of ten for the positive control. 48 hours after challenge exposure all skin examination scores were zero in all test animals.

The skin sensitization potential of the Fatty Acid Polyol with CAS No. 68424-31-7 (Fatty acids, C5-10, esters with pentaerythritol) was evaluated in guinea pigs with a Buehler test for skin sensitisation (Lees, 1991). 20 male albino guinea pigs were treated with the test substance and compared with 10 control animals. Three epidermal inductions were performed with 100 % test substance in weekly intervals for 6 hours under occlusive conditions. 14 days after the last induction treatment, all animals were challenged for 6 hours epicutaneously with 100% (left shorn flank) and 30% (right shorn flank) test substance (diluted in corn oil) under occlusive conditions. Animals were evaluated for skin reactions 24 and 48 h after challenge. No signs for irritation or sensitisation were observed during induction and challenge of the animals.

Migrated from Short description of key information:
Key (test item), Sanders, 2013, not sensitising
Supporting (EC 234-392-1), Blanset, 1997, not sensitising
Supporting (CAS 68424-31-7), Lees, 1991, not sensitising

Justification for selection of skin sensitisation endpoint:
Reliable study (k=1) performed on the substance itself.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Migrated from Short description of key information:
No data on respiratory sensitisation was available.

Justification for classification or non-classification

Based on the results of the mouse local lymph node assay, the substance EC 613 -848 -7 is not classified as sensitising to the skin.